*3.4. Components of the Proteostasis Network*

The proteasome is a complex of multi-subunit proteases, associated with protein degradation, but in protozoan parasites, it has been also involved in cell differentiation and replication processes [50]. In fact, proteasomal inhibitors have been described as promising therapeutic targets for leishmaniasis and trypanosomiasis [51,52]. According to the KEGG database, the complete compendium of proteasomal proteins were identified in this study (Supplementary File, Table S6).

Protein degradation and protein folding cooperate to maintain protein homeostasis or proteostasis [53]. Multiple and drastic environmental changes (pH variation, sudden temperature up-/down-shifts, and oxidative stress) occur along the *Leishmania* life cycle. Most often, these environmental insults promote protein unfolding and aggregation; to counteract these effects, cells possess specialized molecular chaperones (or heat shock proteins: HSPs) that serve as central integrators of protein homeostasis. Not surprisingly, *Leishmania* parasites possess a large number and variety of molecular chaperones [54]. In this study, we identified proteins belonging to the different HSP families: HSP100, HSP83/90, HSP70, HSP60, HSP40/DnaJ, and HSP20 (listed in Supplementary File, Table S7). Mitochondrion is a cellular organelle in which molecular chaperones are of particular relevance because they are involved in protein transport across membranes and protein refolding inside the mitochondria. Recently, the mitochondrial proteome was analyzed in *L. tropica* [22]. Taking advantage of that study, in Table 3, we list those HSPs identified in the *L. infantum* proteome that are potentially mitochondrial proteins.


**Table 3.** Identified molecular chaperones of *L. infantum* promastigotes, with putative mitochondrial location (according to Tasbihi et al. [22]).

*3.5. Glycosomal Proteins Represent a Substantial Fraction of the Experimentally Detected Proteins in the L. infantum Promastigote*

As mentioned above, glycosomes are specialized peroxisomes that contain key enzymes involved on energy metabolism and purine salvage [55]. Moreover, as occurs in peroxisomes, glycosomal proteins are targeted for import to and location in glycosomes by the presence of the peroxisomal targeting signals (PTSs) PTS1 and PTS2. Two essential proteins for targeting newly synthesized proteins, with a PTS2 import signal, into the glycosome are peroxins (PEXs) 5 and 7 [56]. Remarkably, both proteins, PEX5 (LINF\_350019100) and PEX7 (LINF\_290012400), were identified in the experimental proteome of *L. infantum* promastigotes. Moreover, we made a direct comparison between the *L. infantum* proteome reported here and two studies focused on glycosomal proteomes in *Leishmania tarentolae* and *L. donovani* [48,57]. Colasante and collaborators [48] identified 464 proteins in a glycosomal membrane preparation from *L. tarentolae*, and they concluded that 258 would be glycosomal proteins, including 40 enzymes. Interestingly, the orthologs of 165 (64%) of these proteins were experimentally identified in the *L. infantum* promastigote proteome. In particular, complete enzymatic complements involved in glycosomal glycolysis and gluconeogenesis steps were identified in both studies. Jamdhade and coworkers [57] reported the proteome analysis of an enriched glycosome fraction from *L. donovani* promastigotes, identifying 1355 proteins. In our study, orthologs to 853 of those putative *L. donovani* glycosomal proteins were found; these are listed in Supplementary File, Table S8.

The purine salvage pathway, essential for trypanosomes, also takes place in glycosomes [58]. Notably, the 13 enzymes composing the purine salvage pathway (Figure 4) [58] were identified in this *L. infantum* experimental proteome, which was in agreement with the relevance of this metabolic route for parasite survival. In this regard, it is somewhat unexpected that only two enzymes (adenylosuccinate synthetase (ADSS) and inosine monophosphate dehydrogenase (IMPDH)) were identified in the *L. tarentolae* glycosomal proteome, and five of these enzymes were identified in the glycosomal fractions of *L. donovani*. Similarly, we identified most of the enzymes constituting the de novo pyrimidine biosynthesis pathway (Table 4) [59] in the *L. infantum* proteome, whereas Jamdhade et al. [57] only found one enzyme of this pathway in the *L. donovani* glycosomal proteome—the orotate phosphoribosyltransferase (LDBPK\_160560).

**Figure 4.** Enzymes from the purine salvage cycles identified in the *L. infantum*experimental proteome. All the enzymes (grey squares) that are required to complete the pathway wereidentifiedin this study. APRT: adenine phosphoribosyltransferase (LINF\_130016900); NH: nucleoside hydrolase (LINF\_180021400); AK: adenosine kinase (LINF\_300014400); AAH: adenine aminohydrolase (LINF 350026800); ASL: adenylsuccinate lyase (LINF 040009600); AMPDA: AMP deaminase (LINF 130014700); ADSS: adenylosuccinate synthetase (LINF 130016900); GMPR: GMP reductase (LINF 170014800); GMPS: GMP synthase (LINF 220006100); HGPRT: hypoxanthine-guanine phosphoribosyltransferase (LINF 210014900); GDA: guanine deaminase (LINF 290014000); IMPDH: inosine monophosphate dehydrogenase (LINF 190022000); and XPRT: xanthine phosphoribosyltransferase (LINF 210015000). The cycle was depicted according to Boitz et al. [58].


**Table 4.** Enzymesinvolvedin de novo pyrimidine biosynthesisidentifiedin*L.infantum*promastigote proteome.
